Droplet velocity detection

ABSTRACT

Methods and systems are provided for measuring a velocity of a droplet passing through a microfluidic channel.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/932,537, filed Nov. 4, 2015, which claims benefit ofpriority to U.S. Provisional Patent Application No. 62/076,316, filedNov. 6, 2014, which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Microfluidic methods involve passing small volumes of fluid throughmicrofabricated structures and manipulating these volumes to carry outbiological or chemical reactions. To stage such reactions, samples,reactants, solvents, or other reagents can be encapsulated in discretedroplets having volumes on the order of nanoliters or less. A droplet istypically immersed in a carrier fluid from which it is phase-separated,and transported along with the carrier fluid through microfluidicchannels. In sufficiently small channels, this transport occurs at lowReynolds number and exhibits laminar flow. Reactions can be facilitatedby, for example, merging droplets (causing droplet fusion), splittingdroplets (causing droplet fission), injecting material into droplets, orextracting material from droplets.

To control the movement of droplets in a microfluidic device, it can beuseful to measure the velocities of droplets in real time as they passthrough microfluidic channels. Similarly, for droplets subject toinjection or extraction of material, it can be useful to measure thewidths or volumes of these droplets at one or more points in amicrofluidic channel. These measurements can be fed back to systemsgoverning the flow rate of the carrier fluid or the manipulation ofdroplets, allowing optimization of droplet-based reactions. Measuringdroplet velocity or size is challenging, however, because of the smalldimensions of microfluidic devices and the droplets themselves. Imagingindividual droplets with conventional optics requires a high level ofmagnification and a limited field of view. A droplet traveling through amicrofluidic channel at typical velocities can traverse the field ofview faster than two images of the droplet can be acquired inconsecutive video frames. To obtain two or more images of the samedroplet and measure a change, more sophisticated optics can be employedto expand the field of view, or a high-speed camera can be used insteadof a conventional video camera. These solutions are expensive anddifficult to implement.

BRIEF SUMMARY OF THE INVENTION

In a first aspect of the present invention, a method of measuring avelocity of a droplet passing through a microfluidic channel isprovided. The microfluidic channel is interposed between a laser and adetector, and comprises a transparent illumination site. The laser isdirected at the illumination site and the detector. The detectorcomprises a plurality of physically separated detection regions and isconfigured to generate a signal for each region, the signal beingproportional to the intensity of light incident on the region. Themethod includes, while the droplet is absent from the illumination site:shining a laser beam emitted by the laser through the illumination siteand onto the detector, wherein the laser beam is incident on a firstregion and a second region; measuring a first baseline signal for thefirst region, and measuring a second baseline signal for the secondregion. Further, while the droplet passes through the illumination site,the method includes: shining the laser beam through the illuminationsite and onto the detector; measuring a first signal for the firstregion; and measuring a second signal for the second region. The methodalso includes: determining a first departure time at which the firstsignal initially departs from the first baseline signal by a firstpredetermined amount; determining a second departure time at which thesecond signal initially departs from the second baseline signal by asecond predetermined amount; calculating a difference between the firstdeparture time and the second departure time to obtain an elapsed time;and determining a velocity based on the elapsed time, thereby measuringthe velocity of the droplet passing through the microfluidic channel.

In some embodiments of the method, the first region of the detector orthe second region of the detector comprises a single pixel orphotodiode. In some embodiments, determining a velocity comprisesdividing an appropriate distance by the elapsed time. In someembodiments, the first predetermined amount is at least 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80 or 90 percent of the first baseline signal, orthe second predetermined amount is at least 1, 2, 5, 10, 20, 30, 40, 50,60, 70, 80 or 90 percent of the second baseline signal. In someembodiments, the first predetermined amount or the second predeterminedamount is at least 1, 10, 100, 1,000, 10,000, 100,000, or 1,000,000counts. In some embodiments, the first signal initially departs from thefirst baseline signal by falling below the first baseline signal by thefirst predetermined amount, or the second signal initially departs fromthe second baseline signal by falling below the second baseline signalby the second predetermined amount. In some embodiments, the firstsignal initially departs from the first baseline signal by exceeding thefirst baseline signal by the first predetermined amount, or the secondsignal initially departs from the second baseline signal by exceedingthe second baseline signal by the second predetermined amount. In any ofthese embodiments, the first predetermined amount can be about equal tothe second predetermined amount.

The method also includes, in some embodiments, determining a firstrecovery time at which the first signal initially recovers to the firstbaseline signal to within a first predetermined tolerance, the firstrecovery time occurring after the first departure time; calculating adifference between the first departure time and the first recovery timeto obtain a first passage time; and multiplying the first passage timeby the velocity to obtain a width of the droplet. In certainembodiments, the first predetermined tolerance is at most 50, 40, 30,20, 10, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, or 0.01 percent of thefirst baseline signal. In certain embodiments, the first predeterminedtolerance is at most 1,000,000, 100,000, 10,000, 1,000, 100, 10, or 1count(s). The first predetermined tolerance can be about equal to thefirst predetermined amount. In these embodiments, the method can alsoinclude determining a second recovery time at which the second signalinitially recovers to the second baseline signal to within a secondpredetermined tolerance; calculating a difference between the firstrecovery time and the second recovery time to obtain an additionalelapsed time; and determining an additional velocity based on theadditional elapsed time. The first predetermined tolerance can be aboutequal to the second predetermined tolerance.

In some embodiments of the method, determining an additional velocity ofthe droplet comprises dividing an appropriate distance by the additionalelapsed time. In any embodiment, the appropriate distance can be afunction of the distance between the first region and the second regionon the detector.

In a second aspect of the present invention, a method of measuring avelocity of a droplet passing through a microfluidic channel isprovided. The microfluidic channel is interposed between a laser and adetector, and comprises a transparent illumination site. The laser isdirected at the illumination site and the detector. The detectorcomprises a plurality of physically separated detection regions and isconfigured to generate a signal for each region, the signal beingproportional to the intensity of light incident on the region. Themethod includes shining a laser beam emitted by the laser through theillumination site and onto the detector, and identifying a firstnon-incident region and a second non-incident region of the detector,wherein the laser beam is not incident on either non-incident regionwhen the droplet is absent from the illumination site. While the dropletpasses through the illumination site, the method also includes measuringa first signal for the first non-incident region, and measuring a secondsignal for the second non-incident region. Further, the method includesdetermining a first increase time at which the first signal initiallyexceeds a first predetermined threshold; determining a second increasetime at which the second signal initially exceeds a second predeterminedthreshold; calculating a difference between the first increase time andthe second increase time to obtain an elapsed time; and determining avelocity based on the elapsed time, thereby measuring the velocity ofthe droplet passing through the microfluidic channel.

In this aspect, the first non-incident region or the second non-incidentregion can comprise a single pixel or photodiode. In some embodiments,the method further includes, while the droplet is absent from theillumination site, measuring a first dark signal for the firstnon-incident region, and measuring a second dark signal for the secondnon-incident region, wherein the first predetermined threshold is basedon the first dark signal, and the second predetermined threshold isbased on the second dark signal. In these embodiments, the firstpredetermined threshold can be a multiple of at least 1.1, 1.2, 1.5, 2,5, 10, 20, 50, or 100 of the first dark signal, or the secondpredetermined threshold can be a multiple of at least 1.1, 1.2, 1.5, 2,5, 10, 20, 50, or 100 of the second dark signal.

In some embodiments, the first predetermined threshold or the secondpredetermined threshold is at least 1, 10, 100, 1,000, 10,000, 100,000,or 1,000,000 counts. The first predetermined threshold can be aboutequal to the second predetermined threshold. In some embodiments,determining a velocity comprises dividing an appropriate distance by theelapsed time. The appropriate distance can be a function of the distancebetween the first non-incident region and the second non-incident regionof the detector.

In embodiments of the present methods, according to the first or secondaspect of the invention, the appropriate distance can be the width ofthe illumination site, and/or the laser beam can be focused at theillumination site.

In a third aspect of the present invention, a system for measuring thevelocity of a droplet passing through a microfluidic channel isprovided. The system includes a laser, a microfluidic channel, and adetector. The microfluidic channel includes a transparent illuminationsite and is interposed between the laser and the detector. The laser isdirected at the microfluidic channel and the detector, such that a laserbeam emitted by the laser intersects the microfluidic channel at theillumination site and is transmitted by the microfluidic channel to thedetector. The detector comprises a plurality of physically separateddetection regions and is configured to generate a signal for eachregion, the signal being proportional to the intensity of light incidenton the region. In the absence of a droplet at the illumination site, thelaser beam is incident on at least two regions of the detector.

In some embodiments, the system further includes focusing optics,wherein the focusing optics are interposed between the laser and themicrofluidic channel, such that the laser beam is focused at theillumination site. In some embodiments of the system, the detectorincludes at least two non-incident regions, such that the laser beam isnot incident on each non-incident region in the absence of a droplet atthe illumination site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a microfluidic channel imaged with a high speed camera.Droplets are passing through the microfluidic channel. A laser beamtransmitted by the microfluidic channel is incident on the camera.

FIG. 2 shows time-series data for two detection regions of alight-sensitive detector. The detector is a linear photodiode array, andeach detection region is a single element (pixel) in the array. Thecircled portion shows the signals measured for the two regions departingfrom baseline signals.

FIG. 3 shows a laser beam transmitted by a microfluidic channel andincident on a detector. The laser beam intersects the microfluidicchannel at the beam waist and is oriented at an angle with respect tothe microfluidic channel and detector. Geometrical relationships betweenpoints on the microfluidic channel and detector are indicated.

FIG. 4 shows a laser beam focused in a microfluidic channel andilluminating a detector. A droplet is moving through a cross-section ofthe laser beam such that the front of the droplet moves from one edge ofthe beam to the other edge.

FIG. 5 shows a block diagram of a computer system 500 usable withembodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The inventors have discovered that the velocity of a droplet passingthrough a microfluidic channel can be measured by shining a laser beamthrough a transparent portion of the channel and onto a detector. Thedetector comprises a plurality of physically separated detectionregions, and each region can generate a time-series signal that isproportional to the intensity of laser light incident on the region. Inthe absence of a droplet in the transparent portion of the channel, alsocalled the illumination site, the laser beam falls on at least tworegions of the detector. As the droplet passes through the illuminationsite, it can cause the laser beam to be deflected away from theseregions as a result of absorption, scattering, lensing, or refraction.Thus, changes (for example, decreases) in the signals from the two ormore detection regions can be correlated with passage of the dropletthrough the channel. The signal from a first region changes before thesignal from a second region because the droplet takes measurable time totraverse the width of the laser beam in the illumination site, andreaches the portion of the laser beam incident on the first regionbefore it reaches the portion incident on the second region. Bymeasuring the time when the signal from the first region changes by acertain predetermined amount, the time when the signal from the secondregion changes comparably, and the difference between these times, thetime needed for the droplet (or a portion thereof) to travel between twopoints in a cross-section of the laser beam can be determined. Thedistance between these points will be known or can be determined, basedon the size of the illumination site, the geometry of the optical path,the distance between the regions of the detector, or other factors. Thevelocity of the droplet can then be calculated by dividing the distancebetween the two points by the time taken for the droplet to travelbetween them.

The inventors have further found that the velocity of a droplet can becalculated based on the times at which the signals from the twodetection regions recover to their baseline levels, which can berecorded before the droplet enters the illumination site. Changes in thesignals during transit of the droplet across the illumination site canalso be used to calculate the width of the droplet and otherinformation. In addition, droplet velocities can be measured usingsignals from “non-incident” detection regions, which are not in the pathof the laser beam in the absence of a droplet in the illumination site,but receive deflected light as a droplet passes through the illuminationsite. Provided herein are methods and systems for measuring the velocityof the droplet passing through the microfluidic channel.

II. Definitions

“Velocity” refers to the directed rate of movement of an object. In thecase of a microfluidic droplet, velocity can be specified in terms ofthe rate and direction the droplet is moving within a channel (e.g., 10μm/s to the right or 50 μm/s downstream). Velocity can be measured orspecified for the object as a whole, a portion of the object, or a pointon the object, for example the center or the trailing edge. The velocityof an object can be specified in absolute (e.g., 1 mm/s) or relative(e.g., twice as fast as another object) terms, and with respect to anyconvenient reference frame.

“Microfluidic channel” refers to channel or vessel, no more than aboutfive millimeters across in its narrowest dimension, for carrying orholding a fluid.

“Detection region” (equivalently, “detector region” or simply “region”)refers to a portion of a light-sensitive detector, for example a pixelor group of pixels. Alternatively, a detection region can be an entirelight-sensitive detector. Detection regions can be physically separatedfrom each other, meaning that they occur in separate locations from eachother and can receive light originating from different locations.

The words “initial” or “initially”, as used herein, refer to the firstdiscernible time at which an event occurs or a condition is true. Forexample, the time at which signal A initially exceeds signal B is thefirst time at which signal A is measured to be greater than signal B.This time can reflect the resolution of any instruments with which themeasurements are made. An initial time is distinct from later times atwhich the condition remains true, e.g. times at which signal A remainsin excess of signal B.

The terms “about” and “approximately equal” are used herein to modify anumerical value and indicate a defined range around that value. If “X”is the value, “about X” or “approximately equal to X” generallyindicates a value from 0.90X to 1.10X. Any reference to “about X”indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X,1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, “about X” is intended todisclose, e.g., “0.98X.” When “about” is applied to the beginning of anumerical range, it applies to both ends of the range. Thus, “from about6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” isapplied to the first value of a set of values, it applies to all valuesin that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%,about 9%, or about 11%.”

III. Methods

A. Optical and Microfluidic Configuration

According to embodiments of the present invention, methods are providedfor measuring a velocity of a droplet passing through a microfluidicchannel. The microfluidic channel is interposed between a laser and adetector, and includes a transparent illumination site. The laser isdirected at the illumination site and the detector, and is configured toemit a laser beam.

The microfluidic channel employed in the present methods can be of anydimensions and can be part of a larger microfluidic device, such as achip. Suitable materials in which the microfluidic channel can bedisposed, or that can form the walls of the channel, include glass,ceramics, plastics, and polymers. The microfluidic channel can beoptically transparent along some or all of its length so that the laserbeam can be transmitted by the channel to the detector. In someembodiments, all materials immediately surrounding the channel aretransparent. In other embodiments, the microfluidic channel is disposedin an opaque material, with a portion of this material being replacedwith a transparent window or insert to transmit the laser beam. Theillumination site can occur or be designated at any location where themicrofluidic channel is transparent. It will be appreciated thatcomplete transparency of the illumination site is not necessary in allembodiments to achieve acceptable transmission of the laser beam throughthe microfluidic channel, or for the detected laser beam to indicate thepassage of a microfluidic droplet through the microfluidic channel. Insome embodiments, the illumination site is semi-transparent,translucent, or semi-opaque, or transmits different wavelengths of lightwith different efficiencies. For example, the illumination site caninclude a material that selectively passes wavelengths of light emittedby the laser and filters out other wavelengths.

The laser trained on the microfluidic channel and detector can be chosenand configured as desired. Preferably, the laser is operated at a powersuch that the beam does not damage the microfluidic channel, such asthrough heating, ablation, or light-mediated chemical reactions, overthe operational lifetime of the channel. Further, the laser preferablydoes not damage microfluidic droplets, the contents of these droplets,or other fluid species in the channel, or perturb the motion of dropletsthrough trapping forces. Examples of lasers suitable for use in thepresent methods are gas lasers (e.g., helium-neon or argon-ion lasers),solid-state lasers, fiber lasers, and diode lasers. The laser can emitlight in the visible, infrared, or ultraviolet portions of theelectromagnetic spectrum. The laser preferably operates in continuouswave mode, or emits pulses that are of sufficiently short duration orhigh frequency that no measurable movement of the droplet occurs betweenpulses. Such pulses can be several orders of magnitude shorter induration than the time needed for a drop to traverse the illuminationsite or move the width of the laser beam. Suitable pulse durations areon the order of nanoseconds to femtoseconds.

The detector used in the present methods can be selected and configuredas appropriate for the laser. For example, a detector capable ofdetecting red light can be used in conjunction with a 633 nm helium-neonlaser. Similarly, a detector for detecting blue light can be paired witha 488 nm diode laser. The detector includes multiple spatially separateddetection regions that are sensitive to the laser light, and generates asignal for each of these regions that is proportional to the incidentlight intensity. In some embodiments, the detector comprises pixelsarranged in a two-dimensional array, and the regions are individualpixels or groups of pixels. For example, a detection region can include1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, or more pixels. In someembodiments, a detection region includes a single pixel, or at most 1,2, 3, 4, 5, 10, 20, 50, 100, 200, 500, or 1000 pixels. In someembodiments, the detector is a photodiode array (e.g., a linearphotodiode array), and the regions are individual elements (e.g.,photodiodes or pixels) or groups of elements in the array. For example,a detection region can include at least 1, 2, 3, 4, 5, 10, 20, 50, 100,200, 500, or 1000 elements, or at most 1, 2, 3, 4, 5, 10, 20, 50, 100,200, 500, or 1000 elements, in a photodiode array. A region also can bedesignated as a macroscopic portion of an array, or as portion of alight-sensitive surface of the detector. For example, a region can be ahalf or quadrant of this surface. Each region can have any desired shapeor size. For example, a region can be approximately circular (e.g., agroup of pixels arranged in a circle), square, or rectangular, and canhave an average or maximum width of at least 0.01, 0.1, 1, 10, or 100millimeters.

The signals output by the detector for separate detection regions can beindependent of each other or can be coupled, depending on factors suchas the electronic configuration of the detector. If the signals arecoupled, they can be processed to provide separate or approximatelyindependent intensities for the light-sensitive regions. The detectorsignals can be output continuously over time or can be discretelysampled, in which case any desired sampling frequencies, frame rates,exposure times, or integration times can be used. Generally, thedetector provides quantitative time-series information about theintensity of light at each detection region, over timescales that areshort relative to the movement of a droplet through the illuminationsite in the microfluidic channel.

The detector can be part of a video camera or still camera. In someembodiments, the detector is a high-speed camera (FIG. 1). In someembodiments, the detector includes a charge-coupled device (CCD) or acomplementary metal-oxide-semiconductor (CMOS) image sensor.Alternatively, the regions can be photodiodes placed near each other,for example in a linear photodiode array. Variants of and alternativesto these types of image sensors will be apparent to those of skill inthe art.

Signals output by the detector can be processed online (e.g., in realtime) or offline. In the latter case, the signals can be saved to acomputer storage medium for later processing. Signal processing caninclude standard procedures such as smoothing, filtering, sampling, anddigitization. Signal processing can also include quantitativecomparisons between measured signals, or comparisons between a measuredsignal and one or more numeric values, at various time points. In someembodiments, signal processing is performed using a computer system.

The microfluidic channel, laser, and detector can be arranged as desiredaccording to the present methods. For example, any distances can beestablished among the laser, microfluidic channel, and detector. Astraight optical path can be employed, or the laser beam can follow abent or folded path by traveling through an optical fiber or reflectingoff of mirrors, for example. If desired, one or more lenses can beplaced in the optical path to focus or defocus the laser beam, changethe beam profile, or otherwise condition the laser light for betterdetection of droplets passing through the microfluidic channel. Forexample, a pair of lenses forming a telescope or microscope can be usedto set the width of the beam to a desired size at the illumination siteor at the surface of the detector. In some embodiments, one or morelenses are placed immediately after the laser to focus the beam, so thatthe laser beam intersects the microfluidic channel at the beam waist. Insome embodiments, the beam is focused or narrowed at the surface of thedetector so that it is incident on a small area of the detector, forexample a small number of pixels, with this area including two or moredetection regions. Other optics, such as prisms, diffraction gratings,or irises can be used instead or in addition to lenses or mirrors.Optics can be placed between the laser and microfluidic channel, betweenthe microfluidic channel and the detector, or in both locations.

B. Droplet Velocity Measured Using Departure Times

When the laser is turned on, the emitted laser beam intersects themicrofluidic channel at the illumination site and is transmitted by theillumination site to the detector. In the absence of a droplet at theillumination site, the laser beam is incident on at least two regions ofthe detector, i.e. a first region and a second region. These regions canbe adjacent to each other or can be separated. If separated, an unusedportion of the detector (for example, a set of pixels for which nosignals are measured) can occur between the two regions. Alternatively,an optically absorbent or non-reflective (e.g., dark-colored) materialcan be placed between the first region and second region. This materialcan be part of a beam block, beam stop, or beam trap. Other alternativesare to impose an air gap or electrically insulating material between thedetection regions. In preferred embodiments, the detection regions donot overlap. For example, if the detector comprises pixels, the firstregion and second region do not share pixels. Any distance can existbetween two regions or portions thereof. For example, the center pixels,centers, or facing edges of two detection regions can be separated by atleast 0.01, 0.1, 1, 10, or 100 millimeters. If the laser beam isincident on more than two regions, then these regions can be arranged inany desired geometry (for example, in a line or triangle) within thedetector.

In preparation for measuring droplet velocities in the microfluidicchannel, baseline signals are measured for the two detection regions onwhich the laser beam is incident. The baseline signal for a regionindicates the intensity of the portion of the laser beam falling on thatregion when droplets are absent from the illumination site. In someembodiments, the baseline signal is constant or approximately constantover time. A baseline signal can be measured for each region when themicrofluidic channel is filled with the carrier fluid or another fluidhaving a similar index of refraction. For example, if hydrophobicdroplets are carried through the microfluidic channel in an aqueoussolution, then baseline signals can be measured when the channel isfilled with the same aqueous solution, a solution having the samesolutes or solute concentrations as the aqueous solution, or with water.Alternatively, if aqueous droplets are carried through the microfluidicchannel in an oil-based carrier fluid, then baseline signals can bemeasured with the channel containing the same or a similar fluid.Baseline signals can also be measured while droplets are travelingthrough the microfluidic channel, provided the droplets are not withinthe illumination site while the measurements are made. The baselinesignals can be measured before or after droplets pass through theillumination site, and can be measured over any desired time period ornumber of exposures. If desired, a baseline signal can be averaged overtime or exposures to obtain a representative intensity of the laser beamat a particular detection region.

The laser power, detector gain, and/or exposure time are preferablyadjusted to provide, for each detection region, a baseline signal valuethat is near the middle or upper end of the signal's dynamic range.Thus, changes (particularly decreases) in the amount of laser lightincident on the region will register as changes in the value of thesignal, with the amount of light and value of the signal remainingquantitatively correlated. If, on the other hand, the baseline signalfor a region is near the lower end of the dynamic range, then a decreasein the intensity of light incident on the region can cause the signal toflat-line or roll over, making quantification of this decreasedifficult.

While the laser beam is shining through the microfluidic channel andonto the detector, the velocities of one or more droplets passingthrough illumination site can be measured. According to the presentmethods, droplets can be formed, introduced into the microfluidicchannel, and driven through the channel using any desired mechanisms.For example, the channel can be coupled to a pump or pipette tointroduce samples and/or regulate flow. As a droplet passes through theillumination site, the signal for each of the at least two detectionregions is measured and compared with the baseline signal. Preferably,each signal is measured multiple times (e.g., at least 10, 20, 50, 100,200, 500, 1,000, 2,000, 5,000, or 10,000 times), with sufficiently shortmeasurements and times between measurements to observe differentbehaviors in the signals. Each signal is expected to depart from thebaseline signal as the droplet blocks, deflects, or otherwise disturbsthe portion of the laser beam incident on the corresponding detectionregion. Further, the signals are expected to depart from the respectivebaseline signals at different times according to the time needed for thedroplet to cross the width of the laser beam in the illumination site.

The current methods therefore include determining departure times, wherethe departure time for one detection region is the time at which thesignal for that region initially departs, or first differs, from thebaseline signal by a predetermined amount. A first departure time isdetermined for a first region, and a second departure time is determinedfor a second region. If the droplet crosses into the portion of thelaser beam incident on the first region before it crosses into theportion incident on the second region (for example, if the first portionis located upstream of the second portion in the illumination site),then the first departure time will occur before the second departuretime (FIG. 2). Therefore, a difference between the first departure timeand second departure time can be calculated (for example, by subtractingthe first departure time from the second departure time) to obtain anelapsed time. This elapsed time reflects the time needed for the droplet(or a part thereof) to move through the cross-section of the laser beamin the illumination site, between the portions of the beam incident onthe first and second regions of the detector.

Any convenient criteria can be used for selecting the predeterminedamounts used to determine the departure times. The predetermined amountfor the first or second detection region is the amount by which thesignal for that region initially departs from the baseline signal at thefirst or second departure time, respectively. In some embodiments, thefirst predetermined amount is a percentage of the baseline signalmeasured for the first region, or the second predetermined amount is apercentage of the baseline signal measured for the second region. Forexample, the first predetermined amount can be at least 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80 or 90 percent of the baseline signal. In thisexample, at the first departure time, the signal for the first regiondiffers from (exceeds or falls below) the baseline signal by at least 1,2, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 percent. In some embodiments,the first predetermined amount or the second predetermined amount is anumber of counts, for example at least 1, 10, 100, 1,000, 10,000,100,000, or 1,000,000 counts. “Counts” can refer to a number of photonsor other unit of light intensity detected at the detection region duringa certain period, e.g. an exposure or integration time. Thus, the numberof counts registered by the region for such a period, as the dropletpasses through the illumination site, can be compared with the numberregistered for a period of equal or similar duration during measurementof the baseline signal. The first departure time, for example, can occurwhen the number of counts registered for the first region during oneexposure, acquired when the droplet is present in the illumination site,differs from that registered during an exposure when the droplet isabsent. The difference in counts used as the predetermined amount can beset as desired and as appropriate for the detector.

For each of the at least two detection regions of the detector, thesignal can be considered to initially depart from the baseline signalwhen it initially exceeds or falls below the baseline signal by theappropriate predetermined amount. Thus, passage of the droplet throughthe illumination site can be indicated by either an increase or adecrease in the detected signal. The signal detected at one region canincrease if the laser light interacts with the droplet in such a waythat the intensity of light incident on the region is greater than theintensity measured in the absence of the droplet. This can happen, forexample, if the droplet acts as a lens for the laser light, or the laserlight refracts as it passes through the droplet. On the other hand, thesignal detected at a detection region can decrease if the dropletdiverts laser light intersecting the illumination site away from thatregion or diffuses the light. As shown in FIG. 2, the signal for aregion can increase and then decrease in turn as the droplet passesthrough the illumination site. Either the increasing or decreasing phaseof the signal time-series can be used to determine the first or seconddeparture time.

Preferably, similar criteria are used to determine when the signals forthe first and second detection regions initially depart from therespective baseline signals. For example, if the first departure timeoccurs when the signal for the first region falls below the baselinesignal by a predetermined amount (i.e., the signal decreases), then thesecond departure time can occur when the signal for the second regionfalls commensurately. Thus, both the first and second departure timesare based on the signals for the respective detection regionsdecreasing. In some embodiments, the changes in the signals used todetermine the first and second departure times have the same sign ormagnitude. The first predetermined amount can be about equal to thesecond predetermined amount.

Once an elapsed time has been obtained, an uncalibrated or relativevelocity of the droplet can be calculated by taking the reciprocal ofthe elapsed time. The relative velocity can be this reciprocal takenalone, multiplied by a constant, subjected to a linear transformation,or transformed by another appropriate function. The elapsed times orrelative velocities of droplets can be compared to identify changes inthe flow rate in the microfluidic channel. For example, droplets A and Bpass through the illumination site at different times, and the elapsedtime measured for droplet A is half that measured for droplet B. Ifdroplets A and B are assumed to have the same size, then droplet A hastwice the relative velocity of droplet B and is traveling twice as fast.

To determine the absolute velocity for a droplet, an appropriatedistance can be divided by the measured elapsed time. The appropriatedistance corresponds to the distance between the positions in theillumination site where the droplet is transiently located at the firstand second departure times. These positions can occur within across-section of the laser beam in the illumination site, and canrepresent the centroid of the droplet, the leading or trailing edge ofthe droplet, or another part of the droplet.

The appropriate distance can be calculated or estimated as desired. Insome embodiments, the appropriate distance is estimated as the width ofthe laser beam at the illumination site, or the width of theillumination site itself. In some embodiments, the appropriate distanceis a function of the distance between the first and second detectionregions on the detector. This intra-region distance can be determinedwith knowledge of the spacing and geometrical disposition between pixels(e.g., pixels of the same color) on the detector. The spacing can bedisclosed by the manufacturer of the detector or can be measureddirectly using a microscope. The appropriate distance can then becalculated based on the intra-region distance and the geometry of theoptical train (FIG. 3). For example, the intra-region distance can bescaled up or down, as appropriate, to take into account anymagnification of the laser beam occurring between the illumination siteand detector, or any angles between the optical path, microfluidicchannel, and detector, to arrive at the appropriate distance.

C. Droplet Velocity and Width Measured Using Recovery Times

As the droplet exits the illumination site, the droplet no longerperturbs the laser beam or affects the intensity of laser lightregistered at regions of the detector. Accordingly, the signals for thefirst and second detection regions can ‘recover’, or return to theirrespective baseline values. The times at which these signals initiallyrecover provide further information about the droplet and its movementthrough the microfluidic channel. This information can complement thevelocity of the droplet measured as described above, using thedepartures of these signals from the baseline values.

In some embodiments of the present methods, a first recovery time isdetermined. The first recovery time is the time at which the signal forthe first detection region initially recovers to the baseline signal towithin a first predetermined tolerance, and occurs after the firstdeparture time (FIG. 2). The first predetermined tolerance is includedto address the possibility that the signal for the first detectionregion may not fully recover to the baseline signal, within anacceptable amount of time or at all, after the droplet passes throughthe illumination site. A full recovery may not occur due to noise in thesignal for the first region or drift in this signal over time. Inaddition, if multiple droplets are passing through the illumination sitein succession, the signal may not have time to fully recover orstabilize at the baseline value before it departs again, as the nextdroplet enters the illumination site.

Like the first predetermined amount, the first predetermined tolerancecan be selected as desired. In some embodiments, the first predeterminedtolerance is a percentage of the baseline signal measured for the firstdetection region. For example, the first predetermined tolerance can beat most 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, or0.01 percent of the baseline signal measured for the first region. Insome embodiments, the first predetermined tolerance is a number ofcounts as discussed above, for example at most 1,000,000, 100,000,10,000, 1,000, 100, 10, or 1 count(s). The signal for the firstdetection region is considered to be within the first predeterminedtolerance of the baseline signal when the signal differs from thebaseline signal by an amount less than or equal to the firstpredetermined tolerance. As desired, the first predetermined tolerancecan allow for the signal for the first detection region to be largerthan the baseline signal measured for the first region, smaller, orboth. In some embodiments, the first predetermined tolerance is aboutequal to the first predetermined amount.

Once the first recovery time has been determined, a difference betweenthe first departure time and the first recovery time can be calculated(for example, by subtracting the first departure time from the firstrecovery time) to obtain a first passage time. The first passage time isan estimate of the time taken by the droplet (or part of the droplet) topass through the portion of the laser beam that, after exiting theillumination site of the microfluidic channel, is incident on the firstdetection region. It will be recognized that the first passage time issensitive to the first predetermined amount and the first predeterminedtolerance, and may underestimate the transit time of the droplet throughthe portion of the laser beam if these parameters are set too high. Thefirst passage time can be multiplied by the velocity of the droplet toobtain a width of the droplet. For example, if the first passage time is0.01 seconds, and the velocity of the droplet is measured to be 100millimeters per second, then the width of the droplet can be estimatedas 1 millimeter.

In some embodiments, a second recovery time is also determined. Thesecond recovery time is the time at which the signal for the seconddetection region recovers to the baseline signal to within a secondpredetermined tolerance, and occurs after the first recovery time. Thesecond predetermined tolerance is analogous to the first predeterminedtolerance discussed above, but for the second detection region, and alsocan be selected as desired. In some embodiments, the first predeterminedtolerance is about equal to the second predetermined tolerance.

Once the recovery times have been determined, a difference between thefirst recovery time and the second recovery time can be calculated toobtain an additional elapsed time. The result is similar to the elapsedtime calculated using the departure times, and indicates the amount oftime taken by the droplet to pass through a cross-section of the laserbeam in the illumination site. Whereas departure times may indicate whenthe droplet arrives at the portions of the laser beam incident on thefirst and second detection regions, the recovery times may indicate whenthe droplet leaves these portions. Thus, the elapsed time calculatedusing the departure time may represent the position and movement of theleading edge of the droplet, while the additional elapsed timecalculated using the recovery times may represent the position andmovement of the trailing edge of the droplet (FIG. 4). Regardless, theadditional elapsed time can be included in the present methods in thesame way as the elapsed time discussed above. For example, an additionalvelocity can be determined by taking the reciprocal of the additionalelapsed time. The additional velocity can be compared or averaged withthe velocity determined from departure times to obtain a compositevelocity of the droplet.

If desired, the additional velocity can be obtained by dividing anappropriate distance by the additional elapsed time. Here, theappropriate distance corresponds to the distance between the positionsin the illumination site where the droplet is located at the first andsecond recovery times. This appropriate distance can be assumed to bethe same as the distance between the droplet positions at the first andsecond departure times. Thus, the same value for appropriate distance,calculated or estimated as discussed above, can be divided by differentmeasurements of elapsed time to obtain different measurements of dropletvelocity.

D. Droplet Velocity Measured Using Increase Times

The present methods can also involve monitoring regions of the detectorthat are in the path of the laser beam only as a result of droplets inthe illumination site diverting light to these regions. Such regions arecalled “non-incident regions” herein, and are distinct from anydetection regions (e.g., the first region and the second regiondiscussed above) on which the laser beam is incident in the absence of adroplet at the illumination site. In some embodiments, the detectorincludes at least two non-incident regions (i.e., a first non-incidentregion and a second non-incident region). The detector generates asignal for each non-incident region, with the signal being proportionalto the intensity of light incident on the non-incident region. Thesignal, if it rises to a certain level, can indicate a droplet passingthrough the illumination site, and can provide information about thesize and movement of the droplet.

The non-incident regions can occur anywhere on the detector—for example,adjacent to or removed from the first and second regions discussedabove, and with any distances between or among them. In someembodiments, each non-incident region is separated by at least 0.01,0.1, 1, 10, 100, or 1000 millimeters from any detection region on whichthe laser beam is incident in the absence of a droplet at theillumination site. In some embodiments, a first non-incident region anda second-non incident region are at least 0.01, 0.1, 1, 10, 100, or 1000millimeters apart. The non-incident regions can be identified asdesired, for example by monitoring the signals of pixels on the detectorand identifying those undergoing changes as droplets pass through theillumination site. Alternatively, the non-incident regions can bedesignated to detect particular angles or directions of light deflectedby droplets.

To measure the velocity of a droplet using non-incident regions, thepresent methods can include measuring a dark signal for eachnon-incident region when droplets are absent from the illumination site,and while the laser beam is shining. The dark signals are similar to thebaseline signals discussed above in that they represent the intensitiesof light received by regions of the detector while the laser beam is notperturbed by droplets. In the case of non-incident regions, the darksignals represent any ambient light illuminating the detector, plus anysmall amounts of laser light that may reach these regions by randomscattering. Dark signals can be measured while the microfluidic channelis filled with carrier fluid, or while droplets are passing through themicrofluidic channel but remain outside of the illumination site. Thedark signal value for a non-incident region can be measured by averagingthe signal generated for the region over time or over multipleexposures. Preferably, the detector gain and/or exposure time areadjusted to provide, for each non-incident region, a dark signal valuethat is near the bottom of the signal's dynamic range. Thus, thedetector is sensitive to increases in the intensity of light arriving ateach non-incident region.

The methods also include, while the laser beam is shining, measuring asignal for each of the at least two non-incident regions as the dropletpasses through the illumination site. The signals for the non-incidentregions are sampled continuously or repeatedly so that changes in thesesignals over time can be discerned. It is expected that the signals willtransiently increase as the droplet diverts light to the non-incidentregions. Furthermore, it is expected that the signals will increase atdifferent times or by different amounts, because the light diverted toeach of these regions may originate from a different part of thecross-section of the laser beam in the illumination site. The velocityof the droplet can be measured by determining when the signals for thenon-incident regions have exceeded predetermined thresholds, for exampleby comparing these signals to the respective dark signals.

For two of the non-incident regions, increase times are determined. Thefirst increase time is the time at which the signal for a firstnon-incident region initially exceeds a first predetermined threshold.Similarly, the second increase time is the time at which the signal fora second non-incident region initially exceeds a second predeterminedthreshold, where the second increase time occurs after the firstincrease time. If desired, the predetermined thresholds can be set interms of the dark signals for the first and second non-incident regions.For example, in some embodiments, the first (or second) predeterminedthreshold is a multiple of the dark signal measured for the first (orsecond) non-incident region. The multiple can be at least 1.1, 1.2, 1.5,2, 5, 10, 20, 50, or 100, or any other value that represents asignificant increase in the signal above the dark signal. In someembodiments, the first or second predetermined threshold is a number ofcounts, for example 1, 10, 100, 1,000, 10,000, 100,000, or 1,000,000counts. This number can exceed that for the corresponding dark signal byany appropriate amount. In some cases, the predetermined threshold for anon-incident region is set empirically, by observing how the signal forthat region changes as one or more droplets pass through theillumination site. The first predetermined threshold can be about equalto the second predetermined threshold.

An elapsed time for the droplet passing through part of the illuminationsite can then be obtained by calculating a difference between the firstincrease time and the second increase time. This elapsed time representsthe time taken by the droplet to move between two locations in thecross-section of the laser beam, where at each location the dropletdiverts sufficient light to one of the non-incident regions to surpassthe respective predetermined threshold. A velocity can then bedetermined based on the elapsed time, thus yielding a measurement of thevelocity of the droplet. For example, one can take the reciprocal of theelapsed time to obtain an uncalibrated or relative velocity, which canbe compared with relative velocities determined using different methodsand/or for different droplets. If desired, an appropriate distance canbe divided by the elapsed time to determine a velocity. The appropriatedistance in this context can be assumed to be the same as theappropriate distance discussed above. For example, the appropriatedistance can be width of the cross-section of the laser beam at theillumination site, or the width of the illumination site itself.Alternatively, the appropriate distance can be calculated as a functionof the distance between the first non-incident region and the secondnon-incident region on the detector. For example, the intra-regiondistance can be scaled up or down, as appropriate for the geometry oflight traveling between the illumination site and the non-incidentregions of the detector, to obtain the appropriate distance.

E. Comparisons of Velocities

The velocity of a droplet passing through the illumination site of amicrofluidic channel can be measured using any or all of the methodspresented herein. These methods include using departure times, recoverytimes, and increase times. All of these times can be determined for asingle droplet if signals for two or more detection regions on which thelaser beam is incident, as well as two or more non-incident regions, aremonitored during passage. Velocities measured using two or moredifferent methods can be compared, or a composite (e.g., mean) velocitycan be calculated, to characterize or reduce uncertainty in the relativeor absolute velocity of the droplet. The velocities of multiple dropletspassing through the microfluidic channel can also be measured andcompared to determine the flow rate of the solution in which thedroplets are suspended.

IV. Systems

Systems are also provided herein for carrying out the methods describedabove. A system for measuring the velocity of a droplet passing througha microfluidic channel includes a laser, a microfluidic channel, and adetector. These components can have the features discussed above. Forexample, the microfluidic channel can include a transparent illuminationsite and be interposed between the laser and the detector. The laser canbe directed at the microfluidic channel and the detector, such that alaser beam emitted by the laser intersects the microfluidic channel atthe illumination site and is transmitted by the microfluidic channel tothe detector. In the absence of a droplet at the illumination site, thelaser beam can be incident on at least two regions of the detector. Inaddition, the detector can generate a signal for each of these regions,the signal being proportional to the intensity of light incident on theregion.

The components of a system, according to embodiments of the presentinvention, can be positioned and connected as desired. The system canalso include a computer system for performing tasks such as signalprocessing or regulating the rate of flow in the microfluidic channel.

In some embodiments, the system includes a pressure source coupled tothe microfluidic channel. The pressure source can control the speed atwhich droplets and carrier fluid pass through the channel, and, inconjunction with other equipment and fluid sources connected to thechannel, the rate at which droplets increase or decrease in size. Basedupon the velocity of a droplet passing through the microfluidic channel,determined according to the methods described above, the pressure can beadjusted. The result is that the system can accommodate feedback fromthe optical measurements to achieve a target velocity. The pressuresource can be controlled as desired, for example with a computer ormanually. If desired, the pressure source can be controlledautomatically using inputs from the detector.

In some embodiments, the system also includes focusing optics, whereinthe focusing optics are interposed between the laser and themicrofluidic channel, such that a laser beam emitted by the laser isfocused at the illumination site. In some embodiments of the system, thedetector includes at least two non-incident regions, such that the laserbeam is not incident on each non-incident region in the absence of adroplet at the illumination site, and the detector generates a signalfor each non-incident region, the signal being proportional to theintensity of light incident on the non-incident region. Other featuresand elaborations of systems within the scope of the present applicationwill be apparent in view of the disclosure above.

The present disclosure of systems and methods includes droplets passingthrough microfluidic channels. It will be recognized that the methodscan be used to measure velocities or sizes of other objects, and indeedany object that can be illuminated and detected as described herein.

V. Example

A microfluidic channel having a square cross-section (20 μm on eachside) was placed between a 488-nm diode laser and a detector comprisinga linear photodiode array. The laser beam was shone through themicrofluidic channel and onto the detector, such that the beamintersected the microfluidic channel at a transparent illumination siteand was incident on two regions (a first region and a second region) ofthe detector. A suspension of aqueous droplets in fluorinated oil waspassed through the microfluidic channel and signals for the two regionswere monitored in real time. The signals were sampled at 1 MHz, and thenumber of counts registered by each region per 1-μs bin (i.e., perintegration period) was recorded (FIG. 2). A baseline signal of 33counts per bin was measured for the first region, and a baseline signalof 88 counts per bin was measured for the second region. As a dropletpassed through the illumination site, the signal for the first regionwas observed to fall by 27 percent to 24 counts in bin 1050. The signalfor the second region was observed to fall comparably, by 33 percent to59 counts, in bin 1100. The 1050th and 1100th bins were identified asthe first and second departure times, respectively, and an elapsed timeof 50 μs between these bins was calculated. An appropriate distance of10 μm was estimated. The appropriate distance was divided by the elapsedtime to obtain a droplet velocity of 0.20 m/s.

VI. Computer Systems

Any of the computer systems mentioned herein may utilize any suitablenumber of subsystems. Examples of such subsystems are shown in FIG. 5 incomputer apparatus 500. In some embodiments, a computer system includesa single computer apparatus, where the subsystems can be the componentsof the computer apparatus. In other embodiments, a computer system caninclude multiple computer apparatuses, each being a subsystem, withinternal components.

The subsystems shown in FIG. 5 are interconnected via a system bus 575.Additional subsystems such as a printer 574, keyboard 578, storagedevice(s) 579, monitor 576, which is coupled to display adapter 582, andothers are shown. Peripherals and input/output (I/O) devices, whichcouple to I/O controller 571, can be connected to the computer system byany number of means known in the art, such as serial port 577. Forexample, serial port 577 or external interface 581 (e.g. Ethernet,Wi-Fi, etc.) can be used to connect computer system 500 to a wide areanetwork such as the Internet, a mouse input device, or a scanner. Theinterconnection via system bus 575 allows the central processor 573 tocommunicate with each subsystem and to control the execution ofinstructions from system memory 572 or the storage device(s) 579 (e.g.,a fixed disk, such as a hard drive or optical disk), as well as theexchange of information between subsystems. The system memory 572 and/orthe storage device(s) 579 may embody a computer readable medium. Any ofthe data mentioned herein can be output from one component to anothercomponent and can be output to the user.

A computer system can include a plurality of the same components orsubsystems, e.g., connected together by external interface 581 or by aninternal interface. In some embodiments, computer systems, subsystem, orapparatuses can communicate over a network. In such instances, onecomputer can be considered a client and another computer a server, whereeach can be part of a same computer system. A client and a server caneach include multiple systems, subsystems, or components.

It should be understood that any of the embodiments of the presentinvention can be implemented in the form of control logic using hardware(e.g. an application specific integrated circuit or field programmablegate array) and/or using computer software with a generally programmableprocessor in a modular or integrated manner. As user herein, a processorincludes a multi-core processor on a same integrated chip, or multipleprocessing units on a single circuit board or networked. Based on thedisclosure and teachings provided herein, a person of ordinary skill inthe art will know and appreciate other ways and/or methods to implementembodiments of the present invention using hardware and a combination ofhardware and software.

Any of the software components or functions described in thisapplication may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructionsor commands on a computer readable medium for storage and/ortransmission, suitable media include random access memory (RAM), a readonly memory (ROM), a magnetic medium such as a hard-drive or a floppydisk, or an optical medium such as a compact disk (CD) or DVD (digitalversatile disk), flash memory, and the like. The computer readablemedium may be any combination of such storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium according to an embodiment of the presentinvention may be created using a data signal encoded with such programs.Computer readable media encoded with the program code may be packagedwith a compatible device or provided separately from other devices(e.g., via Internet download). Any such computer readable medium mayreside on or within a single computer product (e.g. a hard drive, a CD,or an entire computer system), and may be present on or within differentcomputer products within a system or network. A computer system mayinclude a monitor, printer, or other suitable display for providing anyof the results mentioned herein to a user.

Any of the methods described herein may be totally or partiallyperformed with a computer system including one or more processors, whichcan be configured to perform the steps. Thus, embodiments can bedirected to computer systems configured to perform the steps of any ofthe methods described herein, potentially with different componentsperforming a respective steps or a respective group of steps. Althoughpresented as numbered steps, steps of methods herein can be performed ata same time or in a different order. Additionally, portions of thesesteps may be used with portions of other steps from other methods. Also,all or portions of a step may be optional. Additionally, any of thesteps of any of the methods can be performed with modules, circuits, orother means for performing these steps.

All documents (for example, patents, patent applications, books, journalarticles, or other publications) cited herein are incorporated byreference in their entirety and for all purposes, to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference in its entirety for all purposes. To theextent such documents incorporated by reference contradict thedisclosure contained in the specification, the specification is intendedto supersede and/or take precedence over any contradictory material.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

What is claimed is:
 1. A method of measuring a velocity of a dropletpassing through a microfluidic channel, wherein: the microfluidicchannel is interposed between a laser and a detector, and comprises atransparent illumination site; the laser is directed at the illuminationsite and the detector; and the detector comprises a plurality ofphysically separated detection regions and is configured to generate asignal for each region, the signal being proportional to the intensityof light incident on the region, and the method comprises: while thedroplet is absent from the illumination site, shining a laser beamemitted by the laser through the illumination site and onto thedetector, wherein the laser beam is incident on a first region and asecond region of the detector, measuring a first baseline signal for thefirst region, and measuring a second baseline signal for the secondregion; while the droplet passes through the illumination site, shiningthe laser beam through the illumination site and onto the detector,measuring a first signal for the first region, and measuring a secondsignal for the second region; determining a first departure time atwhich the first signal initially departs from the first baseline signalby a first predetermined amount; determining a second departure time atwhich the second signal initially departs from the second baselinesignal by a second predetermined amount; calculating a differencebetween the first departure time and the second departure time to obtainan elapsed time; and determining a velocity based on the elapsed time,thereby measuring the velocity of the droplet passing through themicrofluidic channel.
 2. The method of claim 1, wherein the first regionof the detector or the second region of the detector comprises a singlepixel or photodiode.
 3. The method of claim 1, wherein: the first signalinitially departs from the first baseline signal by falling below thefirst baseline signal by the first predetermined amount, or the secondsignal initially departs from the second baseline signal by fallingbelow the second baseline signal by the second predetermined amount. 4.The method of claim 1, wherein: the first signal initially departs fromthe first baseline signal by exceeding the first baseline signal by thefirst predetermined amount, or the second signal initially departs fromthe second baseline signal by exceeding the second baseline signal bythe second predetermined amount.
 5. The method of claim 1, wherein thefirst predetermined amount is about equal to the second predeterminedamount.